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1.
The effect of Mg on Ca movement between the sarcoplasmic reticulum (SR) and myofilament space (MFS) was studied in skinned muscle fibers by using isometric force as an indicator of MFS Ca. In Ca-loaded fibers at 20 degrees C, the large force spike induced by Ca in 1 mM Mg (5 mM ATP) was strongly inhibited in 3 mM Mg, and force development was extremely slow. After a brief Ca stimulus in 1 mM Mg, relaxation in Ca-free solution was significantly faster in 3 mM Mg. These changes were due to altered Ca movements, since the effect of 3 mM Mg on steady force in CaEGTA solutions was small. Changes in Mg alone induced force transients apparently due to altered Ca movement. In relaxed fibers, decreasing the Mg to 0.25 mM caused phasic force development. In contracting fibers in Ca solutions, increasing the Mg caused a large transient relaxation. The effects of increased Mg were antagonized by 0.5 mM Cd, an inhibitor of the SR Ca transport system. The results indicate that active Ca uptake by the SR in situ is stimulated by Mg, and that it can affect local MFS [Ca++] in the presence of a substantial Ca source. These results provide evidence that an increased rate of Ca uptake in 3 mM Mg could account for inhibition of the large force spike associated with Ca-induced Ca release in skinned fibers.  相似文献   

2.
Isometric force and 45Ca loss from fiber to bath were measured simultaneously in skinned fibers from frog muscle at 19 degrees C. In unstimulated fibers, 45Ca efflux from the sarcoplasmic reticulum (SR) was very slow, with little or no dependence on EGTA (0.1-5 mM) or Mg++ (20 micrometer-1.3 mM). Stimulation by high [Cl] at 0.11 mM Mg++ caused rapid force transients (duration approximately 10 s) and 45Ca release. This response was followed for 55 s, with 5 mM EGTA added to chelate myofilament space (MFS) Ca either (a) after relaxation, (b) near the peak of the force spike, or (c) before or with the stimulus. When EGTA was present during Cl application, stimulation of 45Ca release was undetectable. Analysis of the time-course of tracer loss during the three protocols showed that when EGTA was absent, 16% of the fiber tracer was released from the SR within approximately 3 s, and 70% of the tracer still in the MFS near the peak of the force spike was subsequently reaccumulated. The results suggest that (a) the Cl response is highly Ca-dependent; (b) stimulation increases 45Ca efflux from the SR at least 100-200-fold; and (c) the rate of reaccumulation is much slower than the influx predicted from published data on resting fibers, raising the possibility that depolarization inhibits active Ca transport by the SR.  相似文献   

3.
The effects of low intracellular free Mg2+ on the myoplasmic calcium removal properties of skeletal muscle were studied in voltage-clamped frog skeletal muscle fibers by analyzing the changes in intracellular calcium and magnesium due to membrane depolarization under various conditions of internal free [Mg2+]. Batches of fibers were internally equilibrated with cut end solutions containing two calcium indicators, antipyrylazo III (AP III) and fura-2, and different concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor [Ca2+] and [Mg2+] transients, whereas fura-2 fluorescence was mostly used to monitor resting [Ca2+]. Shortly after applying an internal solution containing less than 60 microM free Mg2+ to the cut ends of depolarized fibers most of the fibers exhibited spontaneous repetitive movements, suggesting that free internal Mg2+ might affect the activity of the sarcoplasmic reticulum (SR) calcium channels at rest. The spontaneous contractions generally subsided. In polarized fibers the maximal amplitude of the calcium transient elicited by a depolarizing pulse was about the same whatever the internal [Mg2+], but its decay after the end of the pulse slower in low [Mg2+]. In low [Mg2+] (less than 0.14 mM), the mean rate constant of decay obtained from fitting a single exponential plus a constant to the decay of the calcium transients was approximately 30% of its value in the control fibers (1 mM internal [Mg2+]). A model characterizing the main calcium removal properties of a frog skeletal muscle fiber, including the SR pump and the Ca-Mg sites on parvalbumin, was fitted to the decay of the calcium transients. Results of the fits show that in low internal [Mg2+] the slowing of the decay of the calcium transient can be well predicted by both a decreased rate of SR calcium uptake and an expected decreased resting magnesium occupancy of parvalbumin leading to a reduced contribution of parvalbumin to the overall rate of calcium removal. These results are thus consistent with the known properties of parvalbumin as a Ca-Mg buffer and furthermore suggest that in an intact portion of a muscle fiber, the activity of the SR calcium pump can be affected by the level of free Mg2+.  相似文献   

4.
Magnesium effects on activation of skinned fibers from striated muscle   总被引:2,自引:0,他引:2  
The intracellular Ca movements that control contraction and relaxation of striated muscle are regulated by the membrane potential and influenced by Mg2+. In skinned fibers, the internal composition can be manipulated directly by Ca movements estimated from isometric force transients, net changes in sarcoplasmic reticulum (SR) Ca, and 45Ca flux between fiber and bath. Stimulated Ca release, unlike unstimulated 45Ca efflux at low external [Ca2+], is highly [Mg2+]-sensitive at 20 C. Force and tracer measurements indicate three major sites of Mg2+-Ca2+ interaction in situ: Mg2+ can stimulate the SR active Ca transport system, inhibit a Ca2+-dependent Ca efflux pathway of SR, and shift the force-[Ca2+] relation, presumably by reducing Ca2+ binding to myofilament regulatory sites. These mechanisms constrain the resting Ca flux and are adaptive during relaxation. However, analysis of CI-stimulated 45Ca release and reaccumulation suggests that the depolarization process may inhibit Mg2+-dependent Ca influx, the membrane potential controlling both efflux and influx; recent studies on voltage-clamped cut fibers support this hypothesis. The Ca2+ and Mg2+ dependence of caffeine-stimulated 45Ca efflux suggests that Mg2+ inhibition of the Ca2+-dependent efflux pathway is small during rapid Ca2+ efflux. Therefore, both Mg2+ mechanisms, which minimize net release, may be reversed during normal activation.  相似文献   

5.
Rapid Ca2+ release from Ca2+ -loaded sarcoplasmic reticulum vesicles (SR) was previously shown to occur upon the addition of micromolar concentrations of heavy metals, and the extent of Ca2+ release was dependent on the binding affinity of the metal to sulfhydryl group(s) on an SR protein (Abramson, J.J., Weden, L., Trimm, J.L., and Salama, G. (1982) Biophys. J. 37, 134a; Abramson, J.J., Trimm, J.L., Weden, L., and Salama, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1526). The nature of this Ca2+ release site was examined further and found to be predominantly distributed in heavy SR (HSR) rather than light SR fractions. Ag+ -induced Ca2+ release from heavy SR was blocked by local anesthetics and ruthenium red which are known to inhibit Ca2+ release in skeletal fibers and in heavy SR, respectively. The rate of Ca2+ efflux from SR triggered by Ag+ was dependent on pH, Mg2+, and ionic strength of the medium. Efflux rates increased by a factor of 4 from pH 6.0 to 7.0 and then decreased in more alkaline reaction mixtures. Efflux rates from actively or passively loaded SR increased by a factor of 2.5 with increasing Mg2+ from 0 to 1 mM and then decreased in the range of 1 to 10 mM Mg2+. ATP-dependent Ca2+ uptake by SR was similar in 100 mM KCl and in 200 mM sucrose solutions, but the extent and rate of Ca2+ efflux induced by Ag+ were dramatically reduced with decreasing ionic strength of the medium. In solutions containing 5 mM Mg2+, the rate of Ca2+ efflux from heavy SR averaged over the first 1.5 s after the addition of Ag+ was 58 nmol of Ca2+/mg of SR/s, a value comparable to the fast initial rate of ATP-dependent Ca2+ uptake. The maximum initial rate of Ag+ -induced Ca2+ efflux from heavy SR in 1 mM Mg2+ may be comparable to the rate of Ca2+ release and tension development in muscle fibers. Our data indicate that Ag+ reacts with a protein or proteins in the SR, probably not the (Ca2+, Mg2+)-ATPase, to induce a rapid release of Ca2+, possibly from the physiological Ca2+ release site.  相似文献   

6.
We investigated the effect of Cl- on the Ca2+ permeability of rabbit skeletal muscle junctional sarcoplasmic reticulum (SR) using 45Ca2+ fluxes and single channel recordings. In 45Ca2+ efflux experiments, the lumen of the SR was passively loaded with solutions of 150 mM univalent salt containing 5 mM 45Ca2+. Release of 45Ca2+ was measured by rapid filtration in the presence of extravesicular 0.4-0.8 microM free Ca2+ and 150 mM of the same univalent salt loaded into the SR lumen. The rate of release was 5-10 times higher when the univalent salt equilibrated across the SR-contained Cl- (Tris-Cl, choline-Cl, KCl) instead of an organic anion or other halides (gluconate-, methanesulfonate-, acetate-, HEPES-, Br-, I-). Cations (K+, Tris+) could be interchanged without a significant effect on the release rate. To determine whether Cl- stimulated ryanodine receptors, we measured the stimulation of release by ATP (5 mM total) and caffeine (20 mM total) and the inhibition by Mg2+ (0.8 mM estimated free) in Cl(-)-free and Cl(-)-containing solutions. The effects of ATP, caffeine, and Mg2+ were the largest in K-gluconate and Tris-gluconate, intermediate in KCl, and notably poor or absent in choline-Cl and Tris-Cl. Procaine (10 mM) inhibited the caffeine-stimulated release measured in K-gluconate, whereas the Cl- channel blocker clofibric acid (10 mM) but not procaine inhibited the caffeine-insensitive release measured in choline-Cl. Ruthenium red (20 microM) inhibited release in all solutions. In SR fused to planar bilayers we identified a nonselective Cl- channel (PCl: PTris: PCa = 1:0.5:0.3) blocked by ruthenium red and clofibric acid but not by procaine. These conductive and pharmacological properties suggested the channel was likely to mediate Cl(-)-dependent SR Ca2+ release. The absence of a contribution of ryanodine receptors to the Cl(-)-dependent release were indicated by the lack of an effect of Cl- on the open probability of this channel, a complete block by procaine, and a stimulation rather than inhibition by clofibric acid. A plug model of Cl(-)-dependent release, whereby Cl- removed the inhibition of the nonselective channel by large anions, was formulated under the assumption that nonselective channels and ryanodine receptor channels operated separately from each other in the terminal cisternae. The remarkably large contribution of Cl- to the SR Ca2+ permeability suggested that nonselective Cl- channels may control the Ca2+ permeability of the SR in the resting muscle cell.  相似文献   

7.
The role of intracellular free magnesium concentration ([Mg2+]) in modulating calcium release from the sarcoplasmic reticulum (SR) was studied in voltage-clamped frog cut skeletal muscle fibers equilibrated with cut end solutions containing two calcium indicators, fura-2 and antipyrylazo III (AP III), and various concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor calcium transients, whereas fura-2 fluorescence was used to monitor resting calcium. The rate of release (Rrel) of calcium from the SR was calculated from the calcium transient and found to be increased in low internal [Mg2+]. After correcting for effects of calcium depletion from the SR and normalization to SR content, the mean values of the inactivatable and noninactivatable components of Rrel were increased by 163 and 46%, respectively, in low Mg2+. Independent of normalization to SR content, the ratio of inactivatable to noninactivatable components of Rrel was increased in low internal [Mg2+]. Both observations suggest that internal [Mg2+] preferentially modulates the inactivatable component of Rrel, which is thought to be due to calcium-induced calcium release from the SR. This could also explain the observation that, in low internal [Mg2+], the time to the peak of the calcium transient for a 5-ms depolarizing pulse was not very different from the time to the peak of the delta [Ca2+] for a 10-ms pulse of the same amplitude. Finally, in low internal [Mg2+], the calcium transient elicited by a short depolarizing pulse was in some cases clearly followed by a very slow rise of calcium after the end of the pulse. The observed effects of reduced [Mg2+] on calcium release are consistent with a removal of the inhibition that the normal 1 mM myoplasmic [Mg2+] exerts on calcium release in skeletal muscle fibers.  相似文献   

8.
Depolarizing ion gradients stimulate 45Ca release in skeletal muscle fibers skinned by microdissection. Several lines of indirect evidence suggest that sealed transverse (T) tubules rather than sarcoplasmic reticulum (SR) are the locus of such stimulatory depolarization. Two implications of this hypothesis were tested. (a) A requirement for signal transmission was evaluated from the stimulation of 45Ca efflux in fibers that had been highly stretched, an intervention that can impair the electrical stimulation of intact fibers. Length was increased over approximately 95-115 s, after loading with 45Ca and rinsing at normal length; prestimulus 45Ca loss due to stretch itself was very small. In the first study, stimulation of 45Ca release by KCl replacement of K propionate was inhibited completely in fibers stretched to twice slack length, compared with fibers at 1.05-1.1 times slack length. Identical protocols did not alter 45Ca release stimulated by caffeine or Mg2+ reduction, implying that SR Ca release per se was fully functional and inhibition was selective for a preceding step in ionic stimulation. In a second study, stimulation by choline Cl replacement of K methanesulfonate, at constant [K+] [Cl-] product, was inhibited strongly; total 45Ca release decreased 69%, and stimulation above control loss decreased 78%, in segments stretched to twice the length at which sarcomere spacing had been 2.2 micron, compared with paired controls from the same fibers kept at 2.3 micron. (b) Perchlorate potentiation of T tubule activation was evaluated in fibers stimulated at constant [K+] [Cl-] at normal length (2.3 micron); this anion shifts the voltage dependence of intramembrane charge movement and contractile activation in intact fibers. Perchlorate (8 mM) potentiated both submaximal stimulation of Ca2+-dependent 45Ca release by partial choline Cl replacement of K methanesulfonate and the small Ca2+-insensitive 45Ca efflux component stimulated by nearly full replacement in the presence of 5 mM EGTA. These results provide independent support for the hypothesis that the T tubules are the locus of stimulation by depolarizing ion gradients, with junctional transmission of this signal causing SR 45Ca release.  相似文献   

9.
In rectus abdominis muscles of the frog the active shorteningprovoked by 15–40 mM K was supported by Ca, Sr, and Ba,but not by Ni, Co, Mn, Cd, or Zn ions. Addition of the lattercations to a solution containing Ca decreased the responsesin a manner suggesting competitive inhibition. The shorteningof the rectus muscle found in divalent cation-free, low K solutionsis abolished by Ni, Co, Mn, and Mg. In rat muscles a transientincrease in the contractural responses to elevated potassiumwas observed when Ni was applied following partial washout ofCa. In single muscle fibers of the barnacle, development oftension was supported by Ca and Sr, and the other divalent cationswere without effect. Retention of Ca45 in barnacle resting musclefibers soaked in solutions containing 10 mM Ca for 2 min andsubsequently washed for 10 min was 60 ± 3.1 mµMCa/g, whereas retention of Ca45 in contracting muscles similarlyexposed to Ca45 was 156 + 17 mµM Ca/g of fresh muscle.The results are compatible with the idea that activation ofcontraction in some types of muscles is due to entry of extracellularCa.  相似文献   

10.
Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers   总被引:7,自引:4,他引:3       下载免费PDF全文
Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.  相似文献   

11.
Effects of exercise of varying duration on sarcoplasmic reticulum function   总被引:5,自引:0,他引:5  
Sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+-Mg2+-ATPase activity were examined in muscle homogenates and the purified SR fraction of the superficial and deep fibers of the gastrocnemius and vastus muscles of the rat after treadmill runs of 20 or 45 min or to exhaustion (avg time to exhaustion 140 min). Vesicle intactness and cross-contamination of isolated SR were estimated using a calcium ionophore and mitochondrial and sarcolemmal marker enzymes, respectively. Present findings confirm previously reported fiber-type specific depression in the initial rate and maximum capacity of Ca2+ uptake and altered ATPase activity after exercise. Depression of the Ca2+-stimulated ATPase activity of the enzyme was evident after greater than or equal to 20 min of exercise in SR isolated from the deep fibers of these muscles. The lowered ATPase activity was followed by a depression in the initial rate of Ca2+ uptake in both muscle homogenates and isolated SR fractions after greater than or equal to 45 min of exercise. Maximum Ca2+ uptake capacity was lower in isolated SR only after exhaustive exercise. Ca2+ uptake and Ca2+-sensitive ATPase activity were not affected at any duration of exercise in SR isolated from superficial fibers of these muscles; however, the Mg2+-dependent ATPase activity was increased after 45 min and exhaustive exercise bouts. The alterations in SR function could not be attributed to disrupted vesicles or differential contamination in the SR from exercise groups and were reinforced by similar changes in Ca2+ uptake in crude muscle homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

12.
Skinned frog fibers were reversibly activated in Ca-free solutions containing 0 mM KCl, 23 microM free Mg, and having an ionic strength of approximately 50 mM. Contractile force was nearly maximal at 22 degrees - 25 degrees C and decreased at lower temperatures. Maximal force in Ca-free solution at 50 mM ionic strength was close to twice the calcium-activated force with pCa 5 and 190 mM ionic strength. The force in Ca-free solution could be reduced to zero by raising the concentration of free Mg from 23 microM to 1.0 mM at the same ionic strength (50 mM). On stretching the fiber from 2.0 to 3.2 micron the force decreased; this effect was similar to that seen with Ca-activated fiber and the data support the idea that Ca-free tension is made at the cross-bridge level. Isotonic contraction during Ca-free activation showed a velocity transient as in Ca-activated fiber at 190 mM ionic strength, but the transient in the present case was very much prolonged. This finding suggests that contraction mechanisms for force generation and for shortening are essentially the same in the two conditions, but that certain rate constants of cross-bridge turnover are slower for the Ca-free contraction. Also, the results indicate that, in low ionic strength, Ca binding to thin filaments is not essential for unmasking the cross-bridge attachment sites, which suggests that the steric blocking mechanism is modified under these conditions.  相似文献   

13.
Ionic gradients imposed by choline Cl replacement of K methanesulfonate (Mes) at constant [K][Cl] product stimulate 45Ca efflux from skinned muscle fibers; a small, sustained Ca2+-insensitive efflux component, observed in EGTA, appears to grade a much larger Ca2+-dependent component responsible for contractile activation and is likely to reflect intermediate steps in excitation-contraction coupling. The present studies examined ATP-related effects on the Ca2+-insensitive stimulation. 45Ca efflux was measured on segments of frog semitendinosus muscle skinned by microdissection, with isometric force monitored continuously. The Ca2+-insensitive component was potentiated by quercetin, a flavonoid thought to inhibit the sarcoplasmic reticulum (SR) Ca pump by stabilizing a phosphorylated intermediate. Quercetin increased the stimulated net 45Ca release in the absence of EGTA, as expected from inhibition of reaccumulation, but its effectiveness in EGTA indicated potentiation of unidirectional efflux as such. Quercetin also increased unstimulated (control) 45Ca efflux in EGTA, to a smaller extent; potentiation appeared to be a function of efflux, with stimulation above control loss increased approximately 2.6-fold. ATP removal before stimulation, which led to rigor force and increased stiffness, prevented all quercetin effects in EGTA. ATP removal by itself inhibited ionic stimulation of the Ca2+-insensitive component, with little residual increase above the parallel control loss. Addition of the nonhydrolyzable ATP analogue AMP-PCP ([adenylyl-beta,gamma-methylene]diphosphate) (0.8 mM) after ATP removal gave similar results to ATP-free solution, which suggests that adenine nucleotide binding alone does not support stimulation by choline Cl. These results imply a fundamental role for ATP in the excitation of skinned fibers by imposed diffusion potentials; they also suggest that ATP regulates the SR Ca efflux channel, in a manner that could provide the positive feedback in Ca2+-dependent Ca release.  相似文献   

14.
Magnesium transport across sarcoplasmic reticulum (SR) vesicles was investigated in reaction mixtures of various composition using antipyrylazo III or arsenazo I to monitor extravesicular free Mg2+. The half-time of passive Mg2+ efflux from Mg2+-loaded SR was 100 s in 100 mM KCl, 150 S in 100 mM K gluconate, and 370 S in either 100 mM Tris methanesulfonate or 200 mM sucrose solutions. The concentration and time course of Mg2+ released into the medium was also measured during ATP-dependent Ca2+ uptake by SR. In reaction mixtures containing up to 3 mM Mg2+, small changes in free magnesium of 10 microM or less were accurately detected without interference from changes in free Ca2+ of up to 100 microM. Three experimental protocols were used to determine whether the increase of free [Mg2+] in the medium after an addition of ATP was due to Mg2+ dissociated from ATP following ATP hydrolysis or to Mg2+ translocation from inside to outside of the vesicles. 1) In the presence of ATP-regenerating systems which maintained constant ATP to ADP ratios and normal rates of active Ca2+ uptake, the increase of Mg2+ in the medium was negligible. 2) Mg2+ released during ATP-dependent Ca2+ uptake by SR was similar to that observed during ATP hydrolysis catalyzed by apyrase, in the absence of SR. 3) In SR lysed with Triton X-100 such that Ca2+ transport was uncoupled from ATPase activity, the rate and amount of Mg2+ release was greater than that observed during ATP-dependent Ca2+ uptake by intact vesicles. Taken together, the results indicate that passive fluxes of Mg2+ across SR membranes are 10 times faster than those of Ca2+ and that Mg2+ is not counter-transported during active Ca2+ accumulation by SR even in reaction mixtures containing minimal concentrations of membrane permeable ions that could be rapidly exchanged or cotransported with Ca2+ (e.g. K+ or Cl-).  相似文献   

15.
Ca2+ currents (ICa) and myoplasmic Ca2+ transients were simultaneously recorded in single muscle fibers from the semitendinosus muscle of Rana pipiens. The vaseline-gap voltage-clamp technique was used. Ca2+ transients were recorded with the metallochromic indicator dye antipyrylazo III. Ca2+ transients consisted of an early fast rising phase followed by a late slower one. The second phase was increased by experimental maneuvers that enlarged ICa, such as augmenting [Ca2+]o (from 2 to 10 mM) or adding (-)-Bay K 8644 (2 microM). When [Ca2+]o was increased, the second phase of the Ca2+ transients and ICa showed an average increase at 0 mV of 2 +/- 0.9 microM (4) and 1.4 +/- 0.3 mA/ml (4), respectively. (-)-Bay K 8644 increased the late phase of the Ca2+ transients and ICa at 0 mV by 0.8 +/- 0.3 microM (3) and 6.7 +/- 2.0 mA/ml (4), respectively. The initial fast rising phase of the Ca2+ transients was not modified. (-)-Bay K 8644 slowed the time constant of decay of the transients by 57 +/- 6 ms. In other experimental conditions, Ca2+ release from the sarcoplasmic reticulum (SR) was impaired with repetitive stimulation in 1 mM [EGTA]i-containing fibers. Under those circumstances, Ca2+ transients directly followed the time integral of ICa. Pulses to 0 mV caused a large Ca2+ transient that became suppressed when large pulses to 100 mV were applied. In fibers with functioning SR, pulses to 100 mV elicited somewhat smaller or similar amplitude Ca2+ transients when compared with those elicited by pulses to 0 mV. The increase in ICa after raising [Ca2+]o or adding (-)-Bay K 8644 cannot directly explain the change in Ca2+ transients in fibers with functioning SR. On the other hand, when Ca2+ release from the SR is impaired Ca2+ transients depend on ICa.  相似文献   

16.
Major questions in excitation--contraction coupling of fast skeletal muscle concern the mechanism of signal transmission between sarcolemma and sarcoplasmic reticulum (SR), the mechanism of SR Ca release, and operation of the SR active transport system during excitation. Intracellular Ca movement can be studied in skinned muscle fibers with more direct control, analysis of 45Ca flux, and simultaneous isometric force measurements. Ca release can be stimulated by bath Ca2+ itself, ionic "depolarization," Mg2+ reduction, or caffeine. The effectiveness of bath Ca2+ has suggested a possible role for Ca2+ in physiological release, but this response is difficult to analyze and evaluate. Related evidence emerged from analysis of other responses: with all agents studied, stimulation of 45Ca efflux is highly Ca2+-dependent. The presence of a Ca chelator prevents detectable stimulation by ionic "depolarization" or Mg2+ reduction and inhibits the potent caffeine stimulus; inhibition is graded with chelator concentration and caffeine concentration, and is synergistic with inhibition by increased Mg2+. The results indicate that a Ca2+-dependent pathway mediates most or all of stimulated 45Ca efflux in skinned fibers, and has properties compatible with a function in physiological Ca release.  相似文献   

17.
Skinned canine cardiac Purkinje cells were stimulated by regularly repeated microinjection-aspiration sequences that were programmed to simulate the fast initial component of the transsarcolemmal Ca2+ current and the subsequent slow component corresponding to noninactivating Ca2+ channels. The simulated fast component triggered a tension transient through Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR). The simulated slow component did not affect the tension transient during which it was first introduced but it potentiated the subsequent transients. The potentiation was not observed when the SR function had been destroyed by detergent. The potentiation decreased progressively when the slow component was separated by an increasing time interval from the fast component. The potentiation was progressive over several beats under conditions that decreased the rate of Ca2+ accumulation into the SR (deletion of calmodulin from the solutions; a decrease of the temperature from 22 to 12 degrees C). In the presence of a slow component, an increase of frequency caused a positive staircase, and the introduction of an extrasystole caused a postextrasystolic potentiation. There was a negative staircase and no postextrasystolic potentiation in the absence of a slow component. These results can be explained by a time- and Ca2+-dependent functional separation of the release and accumulation processes of the SR, rather than by Ca2+ circulation between anatomically distinct loading and release compartments. The fast initial component of transsarcolemmal Ca2+ current would trigger Ca2+ release, whereas the slow component would load the SR with an amount of Ca2+ available for release during the subsequent tension transients.  相似文献   

18.
A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.  相似文献   

19.
Shmygol A  Wray S 《Cell calcium》2005,37(3):215-223
Release of Ca2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca2+ release is considered to be dependent on the Ca2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca2+ load on the amplitude of [Ca2+]i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca2+ level ([Ca2+]L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca2+ (10 mM) extracellular solution leads to a substantial increase in [Ca2+]L (SR overload). However, despite increased SR Ca2+ content this did not potentiate ATP-induced [Ca2+]i transients. Repetitive applications of ATP in the absence of extracellular Ca2+, as well as prolonged incubation in Ca2+-free solution without agonist, depleted the [Ca2+]L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca2+ release. ATP-induced [Ca2+]i transients were completely abolished when SR Ca2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP3-mediated Ca2+ release on the Ca2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca2+ load below its normal resting level substantially reduces the IP3-mediated Ca2+ release, while Ca2+ overload of the SR has no impact on such release.  相似文献   

20.
In mechanically skinned fibers of the semitendinosus muscle of bullfrogs, we examined the role of membrane sulfhydryl groups on Ca2+ release from the sarcoplasmic reticulum (SR). Hg2+, a sulfhydryl reagent (20-100 microM), induced a repetitive contracture of skinned fibers, and this contracture did not occur in skinned fibers in which the SR had been disrupted by treatment with a detergent (Brij 58). Procaine (10 mM), Mg2+ (5 mM), or dithiothreitol (1 mM) blocked the Hg2+-induced contracture. Ag+ or p-chloromercuribenzenesulfonic acid produced similar contractures to that induced by Hg2+. We conclude that Hg2+ releases Ca2+ from SR of a skinned fiber by modifying sulfhydryl groups on the SR membrane, and suggest that the Ca2+ released by Hg2+ may trigger a greater release of Ca2+ from SR to develop tension.  相似文献   

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